The topography of the active site of acetylcholinesterase from Torpedo californica was probed with the aid of two spin-labeled analogs of the inhibitor decamethonium in which either one or two methyl groups had been replaced by a piperidine ring bearing a nitroxide radical. Both spin labels were better inhibitors of the acetylcholinesterase than decamethonium itself. The nitroxide group of the labels became highly immobilized in the presence of the enzyme. The electron spin resonance of the dinitroxide decamthonium analog indicated that this label bound to acetycholinesterase in an extended conformation.